Patent application title: Method of Analyzing Optical Isomers or Method of Resolving the Same

Abstract:

Provided are a method of quickly and simply confirming the success or
failure of resolution of optical isomers with the use of a column for
resolving optical isomers and a method of simply designing the conditions
of the eluent composition under isocratic elution conditions. In
resolving optical isomers, the success or failure of the resolution can
be simply and quickly confirmed by employing an HPLC gradient elution
analysis method with the use of a column for resolving optical isomers.
When the resolution is successfully conducted, the eluent composition
under isocratic elution conditions can be estimated from the elution time
in the gradient elution analysis.

Claims:

1. A method of analyzing or resolving an optical isomer,
comprising:resolving the optical isomer from a mixture of optical isomers
by a gradient elution method using a solvent of two or more components
and using a solvent-resistant column for resolving an optical isomer for
use in HPLC in which a polysaccharide derivative is used as an asymmetry
recognition agent.

2. A method of analyzing or resolving an optical isomer according to claim
1, wherein the solvent for use in the gradient elution method comprises a
solvent composition of two or more components containing a combination of
a low polar solvent and a high polar solvent; a solvent composition of
two or more components containing a combination of a low polar solvent
and a medium polar solvent; a solvent composition of two or more
components containing a combination of a medium polar solvent and a high
polar solvent; or a solvent composition of three or more components
containing a combination of a low polar solvent, a medium polar solvent,
and a high polar solvent.

3. A method of analyzing or resolving an optical isomer according to claim
2, wherein, to the solvent for use in the gradient elution method, at
least one of an amine compound and an acid compound is/are added in a
proportion of 0.01 to 5.0%.

4. A method of analyzing or resolving an optical isomer, comprising:based
on an elution time of a first peak (t1) obtained by the gradient analysis
of claim 1, resolving an optical isomer under isocratic conditions and at
a composition ratio obtained by multiplying a solvent composition ratio
at the time of t1 calculated from a gradient inclination by a factor
(f1).

5. A method of analyzing or resolving an optical isomer according to claim
4, wherein the factor (f1) is within a range of from 0.8 to 0.1.

6. A method of analyzing or resolving an optical isomer according to claim
5, wherein the factor (f1) is within a range of from 0.5 to 0.1.

7. A method of analyzing or resolving an optical isomer, comprising: in a
case where a compound is eluted after completion of gradient in claim
4,calculating a ratio under isocratic conditions by multiplying a solvent
ratio at the time of completion of gradient by 0.2; andresolving an
optical isomer under isocratic conditions and at a composition ratio
obtained by multiplying a difference between an elution time (t1) of a
first peak and a time (Gt) required for gradient by a factor (f2) to the
ratio.

8. A method of analyzing or resolving an optical isomer according to claim
4, wherein the factor (f2) is within a range of from 4 to 30.

9. A method of analyzing or resolving an optical isomer according to claim
4, wherein the factor (f2) is within a range of from 5 to 20.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a method of analyzing optical
isomers or a method of resolving the optical isomers.

BACKGROUND ART

[0002]Conventionally, in HPLC (high performance liquid chromatography)
analysis, isocratic analysis in which elution occurs with a solvent of a
fixed composition and gradient analysis in which elution occurs while
changing with time a mobile phase composition are employed. In the
isocratic analysis, a stable chromatogram is obtained. However, in the
isocratic analysis, it sometimes takes a long time to resolve components
which are different in the retentivity to a stationary phase, or the peak
may be broadened. In contrast, in the gradient analysis, by changing the
composition ratio of a mobile phase and increasing an elution ability,
analysis time can be shortened and broadening of the peak can be
suppressed.

[0003]Various resolving agents and columns are commercially available as a
stationary phase for resolving optical isomers. As such resolving agents
for optical isomers, used frequently are polysaccharide derivatives which
demonstrates excellent resolution properties in resolving various
compounds. Moreover, in recent years, a solvent-resistant resolving agent
for optical isomers has been developed which can be used under mobile
phase conditions of a polar solvent and, in which isomers a
polysaccharide derivative has been immobilized on a carrier (Patent
Documents 1 and 2). A column for resolving optical isomers, which has
been charged with the polysaccharide-based solvent-resistant resolving
agent, has drawn attention because various solvents has become available
as a mobile phase while high resolution properties of the polysaccharide
derivatives are maintained. However, a trial-and-error procedure is
required for achieving sharp resolution in a short time (preferably 30
minutes or shorter).

Patent Document 1: JP 2751003 A

Patent Document 2: JP 2751004 A

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

[0004]The present invention provides a simple analyzing method or a simple
resolving method using a solvent-resistant column for resolving optical
isomers for use in HPLC in which a polysaccharide derivative is used as
an asymmetry recognition agent.

Means for Solving the Problems

[0005]The inventors of the present invention have extensively studied,
and, as a result, found that analysis conditions for achieving favorable
resolution can be simply obtained by gradient analysis and that
conditions of isocratic analysis can be easily determined from gradient
analysis results. Thus, the present invention has been accomplished.

[0006]That is, the present invention provides a method of analyzing or
resolving an optical isomer, including:

[0007]resolving the optical isomer from a mixture of optical isomers by a
gradient elution method using a solvent of two or more components and
using a solvent-resistant column for resolving an optical isomer for use
in HPLC in which a polysaccharide derivative is used as an asymmetry
recognition agent.

[0008]In addition, the present invention provides a method of analyzing or
resolving an optical isomer, including: based on an elution time of a
first peak (t1) obtained by the gradient analysis of claim 1, resolving
an optical isomer under isocratic conditions and at a composition ratio
obtained by multiplying a solvent composition ratio at the time of t1
calculated from a gradient inclination by a factor (f1).

[0009]According to the present invention, the gradient analysis makes it
possible to rapidly and simply confirm the possibility of asymmetry
recognition by a solvent-resistant column for resolving optical isomers
for use in HPLC in which a polysaccharide derivative is used as an
asymmetry recognition agent. Moreover, conditions of isocratic analysis
or resolution for precise analysis or fraction can be easily determined.

[0021]Hereinafter, the present invention will be described in detail
according to embodiments of the present invention. Commercially-available
HPLC apparatuses can be used.

[0022]A polysaccharide derivative is used as an asymmetry recognition
agent for use in the present invention. Examples of polysaccharide
include cellulose and amylose.

[0023]In the present invention, the polysaccharide derivative refers to a
substance in which the hydroxy group of polysaccharide is modified.
Preferable examples of the polysaccharide derivative include carbamate
derivatives or ester derivatives having an aromatic substituent. Still
more preferable examples thereof include carbamate derivatives or ester
derivatives having an alkylated aromatic group. Particularly preferable
examples of the polysaccharide derivative include amylose
tris(3,5-dimethylphenylcarbamate) and cellulose
tris(3,5-dimethylphenylcarbamate).

[0024]Derivatization of the polysaccharide in the present invention is
performed by known methods. For example, as described in WO 95/23125 and
the like, the polysaccharide derivative can be produced through
dehydration or the like of polysaccharide with a compound which can be
reacted with a hydroxy group of polysaccharide and which includes the
above-mentioned functional group or becomes the above-mentioned
functional group through the reaction with the hydroxy group.

[0025]The solvent-resistant column for resolving optical isomers for use
in HPLC in which the polysaccharide derivative in the present invention
is used as an asymmetry recognition agent refers to a column for HPLC
(high performance liquid chromatography) which has been charged with a
filler for resolving a solvent-resistant optical isomer containing the
polysaccharide derivative.

[0026]Here, the filler for resolving a solvent-resistant optical isomer
containing the polysaccharide derivative refers to a substance in which
the above-mentioned polysaccharide derivative is immobilized on a
carrier. The immobilization can be performed by the methods described in
Patent Documents 1 and 2, and, moreover, JP 2002-148247 A, JP 2004-167343
A, or WO 04/095018.

[0027]The column for HPLC which has been charged with the thus-obtained
filler for resolving a solvent-resistant optical isomer is commercially
available as, for example, Chiral Pack IA, Chiral Pack IB, and the like
from Daicel Chemical Industries, Ltd.

[0028]In gradient analysis, the solvent mixing ratio of a mobile phase,
the solvent composition ratio of a mobile phase, ionic strength, pH, etc.
are changed with time to a convex shape, a linear shape, and a concave
shape. Preferably, the solvent composition ratio of a mobile phase is
changed to a linear shape. The solvent composition for use in a gradient
elution method is a solvent composition of two or more components
containing a combination of a low polar solvent such as hexane and a high
polar solvent (excluding alcohols such as methanol, ethanol, and
2-propanol) such as THF (tetrahydrofuran) and acetone; a solvent
composition of two or more components containing a combination of a low
polar solvent such as hexane and a medium polar solvent such as
chloroform; a solvent composition of two or more components containing a
combination of a medium polar solvent and a high polar solvent; or a
solvent composition of three or more components containing a combination
of a low polar solvent, a medium polar solvent, and a high polar solvent.
To a solvent used for the gradient elution method, either or both of
amine compounds such as diethylamine or/and acid compounds such as
trifluoroacetic acid and acetic acid may be added in a proportion of from
0.01 to 5.0%.

[0029]Next, a method of determining isocratic conditions from gradient
analysis data will be described. First, in the case where a compound is
eluted during the gradient analysis, based on the elution time (t1) of
the first peak obtained by the gradient analysis, the solvent composition
ratio at the time of elution (t1) of the first peak is calculated from
the determined gradient inclination (V). Among the obtained solvent
composition ratios, the ratio of a solvent having a lower ratio under
initial conditions (a higher polar solvent) is multiplied by a factor
(f1) to obtain a composition ratio (C). The isocratic conditions are
determined based on the composition ratio (C) (Equation 1).

[0030]This factor is suitably determined in the range of from 0.8 to 0.1
in view of the elution time of the first peak, a resolution factor, and a
difference between the determined gradient curve and an actual gradient
curve. In order to achieve favorable resolution in a short period of
time, the factor is preferably within the range of from 0.5 to 0.1.

C=(A+V×t1)×f1 (Equation 1)

A: Initial composition ratio(%) of a solvent which is to be subjected to
gradient (a solvent having a lower ratio under initial conditions)V:
Gradient inclination of a solvent (%/min)C: Composition ratio(%) of a
solvent which has been subjected to gradient under isocratic conditions
(a solvent having a lower ratio under initial conditions)

[0031]In contrast, in the case where a compound is eluted after completion
of the gradient, the ratio of a solvent having a lower ratio under
initial conditions among solvent ratios at the time of completion of the
gradient is multiplied by 0.2 to calculate the isocratic ratio (B)
(Equation 3). The isocratic conditions are determined based on the
composition ratio (D) obtained by multiplying a difference between the
elution time (t1) of the first peak and a time (Gt) required for the
gradient by a factor (f2) (Equation 2). The factor (f2) is within the
range of 4 to 30, and preferably within the range of 5 to 20.

D=B+(t1-Gt)×f2 (Equation 2)

B=(A+V×Gt)×0.2 (Equation 3)

[0032]A: Initial composition ratio(%) of a solvent which is to be
subjected to gradient (a solvent having a lower ratio under initial
conditions)

[0033]B: Ratio obtained by converting a solvent composition ratio at the
time of completion of gradient in terms of isocratic conditions

[0034]V: Gradient inclination of a solvent (%/min)

[0035]Gt: Gradient time (min)

[0036]D: Composition ratio(%) of a solvent which has been subjected to
gradient under isocratic conditions (a solvent having a lower ratio under
initial conditions)

EXAMPLES

[0037]Hereinafter, the present invention will be described in more detail
with reference to examples, but is not limited to those examples. HPLC
analysis is performed using a chromatography data processing system:
Chrom Perfect manufactured by Justice Laboratory Softare, a
chromatography control system: BOWIN manufactured by Nippon Bunko Co.,
Ltd., a liquid chromatography pump: PU-1580 manufactured by Nippon Bunko
Co., Ltd., a UV detector for liquid chromatography: PU-1575 manufactured
by Nippon Bunko Co., Ltd., an autosampler for liquid chromatography:
PU-1555 manufactured by Nippon Bunko Co., Ltd., a mixing machine for
liquid chromatography: HG1580-32 manufactured by Nippon Bunko Co., Ltd.,
and a degasser for liquid chromatography: or DG1580-53 manufactured by
Nippon Bunko Co., Ltd.

Example 1

[0038]Using a column manufactured by Daicel Chemical Industries, Ltd.
(Registered trademark: Chiral Pack IA (0.46 cmφ×25 cmL)) as a
column for resolving an optical isomer and using a hexane/THF mixed
solvent as a mobile phase, t-stilbene oxide was divided at a flowrate of
1.0 ml/min at a temperature of 25° C. During the process, analysis
was performed while increasing the THF ratio (v/v) in the mobile phase
from 5% to 95% over 18 minutes. The first peak eluted at 6.30 minutes,
and the second peak eluted at 11.20 minutes. The resolution factor (a)
was 2.48. The THF amount in the mobile phase when the first peak eluted
was about 37% (5%+5%×6.30=36.5%). The ratio was multiplied by 0.2
as a factor (f1) to thereby determine the THF ratio in an isocratic
mobile phase (hexane/THF=93/7), whereby baseline resolution was achieved
in a short time. The results are shown in Table 1 and FIG. 1.

Example 2

[0039]By multiplying 0.33 as a factor (f1) of Example 2 to determine the
THF ratio in an isocratic mobile phase (hexane/THF=88/12), baseline
resolution was achieved in a short time. The results are shown in Table 1
and FIG. 2.

Comparative Example 1

[0040]By multiplying 1 as a factor (f1) of Example 1 to determine the THF
ratio (hexane/THF=63/37), and resolution was performed. Complete
resolution was achieved with difficulty. The results are shown in Table 1
and FIG. 3.

[0041]Using a column manufactured by Daicel Chemical Industries, Ltd.
(Registered trademark: Chiral Pack IA (0.46 cmφ×25 cmL)) as a
column for resolving an optical isomer and using a hexane/THF mixed
solvent as a mobile phase, aminoglutethimide was divided at a flow rate
of 1.0 ml/min at a temperature of 25° C. During the process, the
THF ratio (v/v) in the mobile phase was increased from 5% to 95% over 18
minutes. Dipping was continued while maintaining the composition ratio at
the time of completion of the gradient. The first peak of
aminoglutethimide eluted 20.0 minutes after pouring, and the second peak
eluted 21.78 minutes after pouring. The resolution factor (a) was 1.42.
Based on the results of this gradient analysis, the THF ratio at the time
of the elution of the first peak was converted to the ratio (B) in terms
of isocratic. The ratio (B) was 95×0.2=19. The isocratic conditions
(hexane/THF=68/32) were determined based on the composition ratio (D)
obtained by adding, to the ratio (B), a value obtained by multiplying a
difference between the elution time (t1) of the first peak and a time
(Gt) required for gradient by 6.5 (factor (f2)). Thus, favorable
resolution was achieved. The results are shown in Table 2 and FIG. 4.

Example 4

[0042]In Example 3, the isocratic conditions (hexane/THF=64/36) were
determined based on the composition ratio (D) obtained by adding a value
obtained by multiplying a difference between the elution time (t1) of the
first peak and a time (Gt) required for gradient by 8.5 (factor (f2)).
Thus, favorable resolution was achieved. The results are shown in Table 2
and FIG. 5.

Comparative Example 2

[0043]In Example 3, the isocratic conditions (hexane/THF=72/28) were
determined based on the composition ratio (D) obtained by adding a value
obtained by multiplying a difference between the elution time (t1) of the
first peak and a time (Gt) required for gradient by 4.5 (factor (f2)).
Thus, complete resolution was achieved. The elution time of the first
peak was as long as 25 minutes. The results are shown in Table 2 and FIG.
6.

[0047]In Example 1, using a hexane/ethyl acetate (AcOEt)/diethylamine
(DEA) mixed solvent as a mobile phase, a compound, laudanosine, was
divided. During the process, analysis was performed while fixing the DEA
ratio to 0.1% and increasing the AcOEt ratio (v/v) from 5% to 95% over 18
minutes. As a result, resolution hardly occurred, and the peak eluted at
15.27 minutes. The AcOEt in the mobile phase at the time of the elution
of the peak was expressed by the equation: 5%+5%×15.27=81.35%. The
ratio was multiplied by 0.2 as a factor (f1) to thereby determine the THF
ratio in an isocratic mobile phase (hexane/AcOEt/DEA=84/16/0.1), whereby
partial resolution was achieved. The results are shown in Table 4 and
FIG. 10.

[0048]Resolution was performed by replacing the mobile phase of Example 3
by a hexane/chloroform (CHCl3)/diethylamine (DEA) mixed solvent. During
the process, the CHCl3 ratio (v/v) in the mobile phase was increased from
5% to 95% over 18 minutes. Dipping was continued while maintaining the
composition ratio at the time of completion of gradient. The first peak
of aminoglutethimide eluted 26.68 minutes after pouring, and the second
peak eluted 32.96 minutes after pouring. The resolution factor (a) was
1.27. Based on the results of this gradient analysis, the CHCl3 ratio at
the time of the elution of the first peak was converted to the ratio (B)
in terms of isocratic. The ratio was 95×0.2=19. The isocratic
conditions (hexane/CHCl3/DEA=25/75/0.1) were determined based on the
composition ratio (D) obtained by adding, to the ratio (B), a value
obtained by multiplying a difference between the elution time (t1) of the
first peak and a time (Gt) required for gradient by 6.5 (factor (f2)).
Thus, favorable resolution was achieved. The results are shown in Table 5
and FIG. 11.

[0049]According to the present invention, the success or failure of
resolution can be simply and rapidly confirmed by applying the HPLC
gradient elution analysis using a column for resolving optical isomers
when resolution of optical isomers is performed. Further, when the
resolution is achieved, the eluent composition under isocratic elution
conditions can be easily estimated from the elution time of gradient
elution analysis.